US6775621B1 - Degree of hybridization detection method - Google Patents

Degree of hybridization detection method Download PDF

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Publication number
US6775621B1
US6775621B1 US09/459,712 US45971299A US6775621B1 US 6775621 B1 US6775621 B1 US 6775621B1 US 45971299 A US45971299 A US 45971299A US 6775621 B1 US6775621 B1 US 6775621B1
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amount
probe
probes
sample
hybridization
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Noriko Yurino
Kenji Yamamoto
Toshiaki Ito
Toshimasa Watanabe
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Hitachi Software Engineering Co Ltd
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Hitachi Software Engineering Co Ltd
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Assigned to HITACHI SOFTWARE ENGINEERING CO., LTD. reassignment HITACHI SOFTWARE ENGINEERING CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ITO, TOSHIAKI, WATANABE, TOSHIMASA, YAMAMOTO, KENJI, YURINO, NORIKO
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

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  • the present invention relates to a hybridization detection method for the analysis of the presence or absence of a sequence of interest in a biopolymer sample by utilizing the hybridization between the sample biopolymer and a probe biopolymer, and also relates to a biochip applicable for the method.
  • hybridization methods have been often utilized which uses a nucleic acid or protein having a known sequence as a probe.
  • a sample DNA labeled with a fluorescent material is hybridized to a probe DNA immobilized onto a substrate.
  • the sample DNA is bound to the probe DNA, the sample DNA is in turn immobilized on the substrate together with the probe DNA.
  • the fluorescent material attached to the sample DNA is fluorescently excited by irradiation with excitation light from a light source to emit fluorescence, and the fluorescence is then detected. In this manner, the hybridization between the sample DNA and the probe DNA can be detected.
  • FIGS. 7, 8 and 9 The principle of the prior art hybridization detection method as described above is illustrated in FIGS. 7, 8 and 9 .
  • a given amount of probe DNA 1 a is immobilized on a substrate (e.g., a glass plate) 4 as a spot 3 a .
  • Another probe DNAs 1 b , 1 c , . . . are also immobilized on the substrate 4 as spots 3 b , 3 c , . . . , respectively. In this case, however, it is impossible to immobilize all of the probe DNAs in equal amounts on the respective spots.
  • each of sample DNAs 5 a , 5 b , 5 c , . . . is labeled with a fluorescent material 6 .
  • the substrate 4 spotted with the probe DNAs 5 a , 5 b , 5 c , . . . is placed in a hybridization solution 7 and then the fluorescently labeled sample DNAs 5 a , 5 b , 5 c , . . . are added thereto to cause the hybridization between the probe DNAs and the sample DNAs.
  • the hybridization solution 7 is a mixed solution comprising, for example, formaldehyde, SSC (sodium chloride/trisodium citrate), SDS (sodium dodecyl sulfate), EDTA (ethylenediaminetetraacetic acid), distilled water, and the like, in which the mixing ratio between the components may vary depending on the nature of the DNAs employed.
  • SSC sodium chloride/trisodium citrate
  • SDS sodium dodecyl sulfate
  • EDTA ethylenediaminetetraacetic acid
  • the sample DNA is hybridized to the probe DNA to form a double-stranded structure (see the illustrations for the probe DNAs 1 a and 1 b ). If not, the sample DNA remains unbound (see the illustration for the probe DNA 1 c ). As illustrated in FIG. 8C, if any of the sample DNAs is complementary to any of the probe DNAs, the sample DNA is hybridized to the probe DNA to form a double-stranded structure (see the illustrations for the probe DNAs 1 a and 1 b ). If not, the sample DNA remains unbound (see the illustration for the probe DNA 1 c ). As illustrated in FIG.
  • the detection of the hybridization can be performed by irradiating the substrate 4 with excitation light from a lamp 9 (i.e., an excitation light source) to excite the fluorescent material 6 , cutting off the light having wavelengths out of the emission wavelength range of the fluorescent material 6 with an optical filter 10 , and then detecting the light emitted from each spot with a two-dimensional photosensor 8 (e.g., a CCD camera).
  • a lamp 9 i.e., an excitation light source
  • the fluorescent material 6 is present, and therefore fluorescent emission can be detected by exciting the fluorescent material 6 by irradiation with excitation light from the lamp 9 .
  • the spots where the hybridization does not take place e.g., spot 3 c
  • no fluorescent material 6 is present, and therefore no fluorescent emission is observed by irradiation with excitation light from the lamp 9 .
  • a light or dark spot is observed depending on the presence or absence of the hybridization event.
  • the image data detected by the two-dimensional photosensor 8 is transferred to a computer 13 via a controller 12 and indicated on a display.
  • the object of the present invention is to provide a detection method which can quantitatively determine the degree of the hybridization between a probe DNA and a sample DNA.
  • a probe biopolymer and a sample biopolymer are labeled with different fluorescent materials, so that the probe biopolymer and the sample biopolymer present on spots deposited on a substrate can be detected separately for each spot utilizing the difference of the emission wavelength of the fluorescent materials.
  • the emission wavelength of the fluorescent material labeling the probe biopolymer and the emission wavelength of the fluorescent material labeling the sample biopolymer are detected separately, so that it becomes possible to separately detect and, therefore, quantitatively determine the amount of the probe biopolymer and the amount of the sample biopolymer hybridized to the probe biopolymer for each spot.
  • a fluorescent material that has labeled a probe biopolymer is caused to emit fluorescence to determine the amount of the probe biopolymer immobilized on a spot deposited on a substrate (e.g., a glass plate).
  • a substrate e.g., a glass plate
  • Another type of fluorescent material that has labeled a sample biopolymer is also caused to emit fluorescence to determine the amount of the sample biopolymer hybridized to the probe biopolymer.
  • the difference between the amount of the probe biopolymer and the amount of the sample biopolymer is normalized with the amount of the probe biopolymer. Based on the normalized value, the amount of the sample biopolymer relative to the amount of the probe biopolymer spotted on the substrate can be determined.
  • biopolymer refers to any polymeric material constituting a living body, such as DNA, RNA and a protein.
  • one aspect of the present invention is a hybridization detection method for detecting the hybridization between a probe and a sample, which comprising detecting both the amount of the probe and the amount of the sample bound to the probe.
  • probe refers to any biopolymer to be immobilized onto a substrate, such as DNA
  • sample refers to any biopolymer to be hybridized to the probe, such as DNA.
  • Another aspect of the present invention is a hybridization detection method for detecting the hybridization between a probe and a sample, which comprising detecting a value produced by normalizing the difference between the amount of the probe and the amount of the sample bound to the probe with the amount of the probe.
  • the amount of the probe may be detected prior to the hybridization, while the amount of the sample bound to the probe may be detected after the completion of the hybridization. Alternatively, both the amounts of the probe and the sample bound to the probe may be detected after the completion of the hybridization.
  • the detection of the amounts of the probe and the sample bound to the probe may be performed by labeling the probe and the sample with different labeling materials and then detecting the labeling materials separately.
  • a value produced by normalizing the difference between the amount of the probe and the amount of the sample bound to the probe with the amount of the probe may be indicated on a display.
  • the probe is immobilized on a support.
  • the support comprising the probe is a biochip.
  • Still another aspect of the present invention is a biochip comprising a fluorescently labeled probe spotted on a substrate.
  • the amount of the probe immobilized onto a substrate may be different for each probe and each substrate.
  • a biochip 1 has 10 ng of a probe immobilized thereon, while a biochip 2 has 8 ng of the same type of probe immobilized thereon; the fluorescent intensities of the probe before the hybridization are 100 for the biochip 1 and 80 for the biochip 2 in terms of a 256-level gradation; and when the samples A and B are hybridized to the probe on the biochips 1 and 2 , respectively, the fluorescent intensities of the samples are 70 for the biochip 1 (sample A) and 60 for the biochip 1 (sample B). From these result, it would be assessed that the DNA amount of the sample A hybridized to the probe is larger than that of the sample B hybridized to the probe.
  • the ratio of the DNA amount of each sample hybridized to the probe can be calculated by determining the difference between the DNA amount of the probe initially immobilized onto each biochip and the DNA amount of the sample bound to the probe and then normalizing the obtained difference with the DNA amount of the probe, as follows:
  • the method of the present invention enables a more precise analysis of the hybridization compared with the prior art methods in which the hybridization is analyzed only based on the fluorescent intensity of a sample after the hybridization.
  • FIG. 1 illustrates the principle of an embodiment of the hybridization detection method in accordance with the present invention.
  • FIGS. 2A, 2 B and 2 C also illustrate the principle of the embodiment of the hybridization detection method in accordance with the present invention.
  • FIG. 3 also illustrates the principle of the embodiment of the hybridization detection method in accordance with the present invention.
  • FIG. 4 is a flow chart of the processing steps for providing an evaluation value for hybridization of a sample to a probe in the hybridization detection method in accordance with the present invention.
  • FIGS. 5A and 5B illustrate the principle of another embodiment of the hybridization detection method in accordance with the present invention.
  • FIGS. 6A and 6B also illustrate the principle of the another embodiment of the hybridization detection method in accordance with the present invention.
  • FIG. 7 illustrates the principle of an embodiment of the hybridization detection method in accordance with the prior art.
  • FIGS. 8A, 8 B and 8 C also illustrate the principle of the embodiment of the hybridization detection method in accordance with the prior art.
  • FIG. 9 also illustrates the principle of the embodiment of the hybridization detection method in accordance with the prior art.
  • probe biopolymers and sample biopolymers are DNAs.
  • other biopolymers such as RNAs and proteins are also applicable in the present invention.
  • FIGS. 1 to 3 illustrate the principle of an embodiment of the hybridization detection method in accordance with the present invention.
  • each of probe DNAs 1 a , 1 b , 1 c , . . . are labeled with the same fluorescent material 2 .
  • the fluorescent material 2 for example, fluoresce in isothiocyanate (FITC) may be used.
  • the probe DNA 1 a is immobilized onto a glass plate (substrate) 4 as a spot 3 a .
  • Other probe DNAs 1 b , 1 c , . . . are also immobilized onto the glass plate 4 as spot 3 b , 3 c , . . . , respectively.
  • each of sample DNAs 5 a , 5 b , 5 c , . . . is labeled with another type of fluorescent material 6 .
  • the fluorescent material 6 for example, Cy 5 may be used.
  • the glass plate 4 onto which the probe DNAs are spotted is placed in a hybridization solution 7 and the fluorescently labeled sample DNAs are added thereto.
  • the hybridization solution 7 is a mixed solution comprising, for example, formaldehyde, SSC (sodium chloride/trisodium citrate), SDS (sodium dodecyl sulfate), EDTA (ethylenediaminetetraacetic acid) and distilled water, in which the proportion of the components may vary depending on the nature of the probe DNAs and sample DNAs employed.
  • SSC sodium chloride/trisodium citrate
  • SDS sodium dodecyl sulfate
  • EDTA ethylenediaminetetraacetic acid
  • the sample DNA is hybridized to the probe DNA to form a double-stranded structure, as illustrated in FIG. 2C (see the illustrations for the sample DNAs 5 a and 5 b and the probe DNAs 1 a and 1 b ).
  • the sample DNA remains unbound, as illustrated in FIG. 2C (see the illustration for the probe DNA 1 c ). That is, on the spots in which the hybridization takes place (e.g., spots 3 a and 3 b ), both the fluorescent material 2 labeling the probe DNAs and the fluorescent material 6 labeling the sample DNAs individually bound to the probe DNAs are present. In contrast, on the spots in which no hybridization takes place (e.g., 3 c ), only the fluorescent material 2 labeling the probe DNAs is present.
  • the detection of the hybridization may be performed in the following manner, as illustrated in FIG. 3 .
  • the fluorescent material 6 labeling the sample DNAs, and the fluorescent material 2 labeling the probe DNAs are excited by irradiation with excitation light from a lamp 9 to emit fluorescence.
  • the lamp 9 used as an excitation light source may be, for example, a xenon lamp having the emission wavelength range from about 300 to about 700 nm. The reason why such lamp is used is that it can cause the fluorescent emission of both FITC (excitation wavelength: 490 nm, emission wavelength: 520 nm) and Cy 5 (excitation wavelength: 650 nm, emission wavelength: 667 nm).
  • reading of emission from FITC and Cy 5 is taken by using a two-dimensional photosensor 8 through an optical filter 10 having transmission wavelength of 520 nm for FITC and through another optical filter 11 having transmission wavelength of 667 nm for Cy 5 .
  • the date from the two-dimensional photosensor is transferred to a computer 13 via a controller 12 .
  • the two-dimensional photosensor 8 may be, for example, a CCD camera.
  • the optical filters 10 and 11 are movable in the direction of the arrow by driving a stage 14 , and changeable to each other.
  • the amount of each probe DNA is calculated for each spot based on the emission reading from FITC, and then the amount of the sample DNA hybridized to the probe DNA is calculated for each spot based on the emission reading from Cy 5 .
  • a smaller Ci value means that the amount of a sample DNA hybridized to a probe DNA is larger, which also means the sample DNA has higher complementarity to the probe DNA.
  • FIG. 4 is a flow chart of the processing steps for providing an evaluation value Ci.
  • step 11 the position number of a spot to be calculated for the evaluation value is preset.
  • step 12 both the emission quantity Ai of FITC and the emission quantity Bi of Cy 5 are determined for a spot i.
  • the evaluation value Ci thus obtained represents the amount of the probe DNA not hybridized with any sample DNA. Based on the evaluation value, the degree of the complementarity between the sample DNA and the probe DNA can be assessed.
  • step 14 the obtained evaluation value Ci is indicated on a display of the computer 13 as a tone image.
  • step 15 it is checked whether processing of all of the spots to be examined is completed. If not, the position number of a spot to be processed in the next step is determined in step 16 , and the procedures from step 12 to step 15 are repeated. When the calculation for all of the spots to be examined is completed, the process exits.
  • FIGS. 5 and 6 illustrate the principle of another embodiment of the detection method in accordance with the present invention.
  • the detection method of this embodiment includes the steps of reading an emission quantity of a fluorescent material (e.g., FITC) labeling a probe DNA, which corresponds to the amount of a probe DNA, prior to the hybridization; reading an emission quantity of another fluorescent material (e.g., Cy 5 ) labeling a sample DNA, which corresponds to the amount of a sample DNA, after the completion of the hybridization; and then determining an evaluation value as described above.
  • a fluorescent material e.g., FITC
  • another fluorescent material e.g., Cy 5
  • probe DNAs each labeled with a fluorescent material FITC are immobilized onto a glass plate (a substrate) 4 as spots 3 a , 3 b , 3 c , . . . .
  • the immobilization of the probe DNAs may be performed in the same manner as illustrated in FIG. 1 .
  • FITC labeling each probe DNA is exited by irradiation with excitation light to emit fluorescence, and the emission quantity of FITC is read with a two-dimensional photosensor 8 , thereby determining the amount of each of the probe DNAs present for each spot.
  • an optical filter 10 having transmission wavelength of 520 nm is located in the optical path of the photo sensor 8 .
  • the reading of the emission quantity may be performed in the same manner as illustrated for FITC in FIG. 3 above.
  • the emission quantity Ai for each spot is stored in a storage medium 14 (e.g., a floppy disk).
  • the hybridization between sample DNAs 5 a , 5 b , . . . each labeled with a fluorescent material Cy 5 6 and the probe DNAs may be performed in the same manner as illustrated in FIG. 2B above.
  • the detection of the hybridization may be performed, as illustrated in FIG. 6B, by irradiating the glass plate 4 with excitation light from the lamp 9 to cause to emit fluorescence from Cy 5 , and reading the emission quantity Bi of Cy 5 with the two-dimensional photosensor 8 .
  • another optical filter 11 having transmission wavelength of 667 nm is located in the optical path of the photosensor 8 .
  • the reading of the emission quantity may be performed in the same manner as illustrated for Cy 5 in FIG. 3 above.
  • the emission quantity Ai of FITC stored in the storage medium 14 is then inserted into the computer 12 , and the difference between the emission quantity Ai of FITC and the emission quantity Bi of Cy 5 is determined. Each of the determined difference values is divided by the emission quantity Ai of FITC. The determination of the difference may be performed in the same manner as illustrated in FIG. 3 above. The method of this embodiment also enables to quantitatively determine the amounts of a sample DNA hybridized to a probe DNA.
  • the method of this embodiment enables to perform a hybridization experiment with a good efficiency due to its capacity to address such problems prior to the experiment.
  • the present invention it becomes possible to determine the amount of a probe spotted on a substrate and the amount of a sample hybridized to the probe; to calculate the precise amount of the sample hybridized to the probe (i.e., the degree of the complementarity) by determining a value produced by normalizing the difference between the amount of the probe and the amount of the sample with the amount of the probe; and therefore to determine the amount of the sample bound to the probe with a high precision.

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Cited By (3)

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US20050014147A1 (en) * 2001-08-21 2005-01-20 Hessner Martin J Method and apparatus for three label microarrays
EP1975247A1 (de) * 2007-03-30 2008-10-01 Micronas GmbH Biochip und Verfahren zu dessen Herstellung
US20090305426A1 (en) * 2005-02-16 2009-12-10 Life Technologies Corporation Axial illumination for capillary electrophoresis

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JP2003028797A (ja) * 2001-07-11 2003-01-29 Hitachi Software Eng Co Ltd 蛍光読み取り装置
DE10145701A1 (de) * 2001-09-17 2003-04-10 Infineon Technologies Ag Fluoreszenz-Biosensorchip und Fluoreszenz-Biosensorchip-Anordnung
WO2004017068A1 (ja) * 2002-08-12 2004-02-26 Hitachi High-Technologies Corporation Ｄｎａマイクロアレイを用いた核酸検出方法及び核酸検出装置

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050014147A1 (en) * 2001-08-21 2005-01-20 Hessner Martin J Method and apparatus for three label microarrays
US20090305426A1 (en) * 2005-02-16 2009-12-10 Life Technologies Corporation Axial illumination for capillary electrophoresis
US20110143446A1 (en) * 2005-02-16 2011-06-16 Life Technologies Corporation Axial Illumination for Capillary Electrophoresis
US8446588B2 (en) 2005-02-16 2013-05-21 Applied Biosystems, Llc Axial illumination for capillary electrophoresis
US9285316B2 (en) 2005-02-16 2016-03-15 Applied Biosystems, Llc Axial illumination for capillary electrophoresis
EP1975247A1 (de) * 2007-03-30 2008-10-01 Micronas GmbH Biochip und Verfahren zu dessen Herstellung

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DE69930826D1 (de) 2006-05-24
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EP1010764A3 (de) 2003-01-22
DE69930826T8 (de) 2007-08-23
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US20040161801A1 (en) 2004-08-19
JP3442327B2 (ja) 2003-09-02

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